Investigations of hematopoietic growth factor bio-effects and action mechanisms continue to provide important insight into (dys) regulated blood cell formation. The EPO receptor (EPOR) system is an informative and clinically significant paradigm. Recent studies applying contemporary approaches indicate major gaps in the field's understanding of EPO/EPOR/JAK2 signal transducers and their regulation of erythroid progenitor cell (EPC) formation. To illustrate, the PI has recently identified a novel EPOR/JAK2/STAT5 pathway in which an EPO-induced Spi2A serpin cytoprotects EPCs against executioner cathepsins as leached from ROS-compromised lysosomes [JEM 210:225-32]. And Dr. T. Ganz's laboratory has characterized an EPOR/JAK2/STAT5-induced Erythroferrone TNF cytokine as a hepcidin suppressor [Nat Genet. 46:678-84]. Via major supporting studies for this R01 renewal, we've applied post-translational modification (PTM) based LC-MS/MS proteomics to discover intriguing new mediators of EPO/EPOR/JAK2 action. These include 50+ factors not previously linked to EPO-dependent erythropoiesis within diverse functional categories of molecular adaptors, erythroid cytoskeletal proteins, kinases & phosphatases, and cell cycle & survival factors. SA#1 will extend our PTM-directed proteomic investigations in human erythroid precursor cells to include broad-based targets as modified at pY, T*PP and ubiquitin motifs, together with analyses of more select signaling nodes for S/T kinases, survival/apoptosis factors and cell cycle targets. Networks for hundreds of specifically activated PTM events for novel (and known) EPO targets and transducers will be mined (with collaborating expert bioinformaticists). SA#2 focuses on defining the functional roles and action mechanisms of three interrelated new EPO targets as upstream effectors of EPOR/JAK2 complexes. Two, C1ORF186/RHEX and C1ORF150, are novel molecular adaptors that have evolved as EPO signal transducers in hEPC's (but are absent among mice, rats, lower vertebrates). Third, PTPN18 is a protein tyrosine phosphatase which we demonstrate to increase JAK2 activation, decrease EPOR turnover and limit pY-RHEX formation. Approaches will include GOF, shRNA LOF, and mutant rescue studies in UT7epo cells and primary hEPCs. SA#3 then focuses on a new downstream mediator of EPO action, Thioredoxin-Interacting Protein (TXNIP). EPO modulates TXNIP at C-terminal pT/pS sites, and heightens its expression. TXNIP knockdown attenuates EPC growth, and notably accelerates primary erythroid precursor development to KIT-low, GPA-high hemoglobinizing erythroblasts. Mechanistically how TXNIP acts as a novel EPO agent will be determined by analyzing EPC growth, survival, ROS, miRNA populations and metabolic properties. Overall, studies will reveal important new mediators of EPO-dependent human erythropoiesis. Certain may be druggable with potentials to lessen EPO dosing, and limit EPO's major adverse side effects. Other new EPO targets may functionally relate to MPNs and/or to EPO's potential to worsen cancer outcomes.